The lack of effective treatments for mutant RAS driven cancers remains a critical unmet need in modern cancer therapy. Several lines of recent evidence suggest that Kinase Suppressor of RAS (KSR) is a viable target for small molecule therapeutics. It has been established that KSR binds constitutively to MEK and transiently to RAF and Erk during active RAS signaling, and these binding events are all mediated at least in part through KSR's protein kinase domain. Recent structural studies by Wellspring's scientific co-founder, Kevan Shokat suggest that KSR's kinase domain functions as a bifunctional dynamic scaffold, bringing together key proteins for RAS signaling (MEK and RAF) and allosterically modulating RAF mediated MEK phosphorylation. A generic kinase inhibitor with the ability to modulate the allosteric switch in KSR was recently reported by Shokat and coworkers, suggesting a possible starting point to develop novel drugs that function as chemical suppressors of Ras via KSR. We are proposing to leverage the structural and biochemical findings of Shokat and colleagues, coupled with an established kinase chemical biology strategy, to generate small molecule binders of the KSR ATP site and determine their impact on KSR supported RAS signaling and cellular transformation. Our hypothesis is that binding to the KSR ATP-site will interfere with allosteric transitions required for KSR mediated RAS signaling. If successful, we will have developed a novel therapeutic strategy for the treatment of RAS driven tumors. The Phase I specific aims are: (1) Validate the ability of ATP-site directed small molecules to block KSR function (2) Develop assays for profiling KSR ATP- site occupancy. In collaboration with the Arvin Dar lab, the work outlined in this Phase I SBIR grant will provide a foundation for compound screening and characterization efforts, and expand our knowledge of the biochemical requirements for KSR mediated RAF-MEK phosphorylation to help guide compound design strategy. Subsequent Phase II studies would be aimed at identification, optimization, and characterization of reversible inhibitors of KSR and the selection of a candidate for progression into clinical development. The successful development of small molecules capable of disrupting KSR supported mutant RAS signaling would represent a major milestone in the treatment of human cancers.